Chromosome abnormalities such as deletions, multiplication of chromosomal segments and gene amplifications are often associated with cancers. The amplification and deletion of DNA sequences containing proto-oncogenes and tumor suppressor genes are characteristic of tumorigenesis. In breast tumors, for example, comparative genomic hybridization has revealed approximately 20 regions of recurrent increased DNA copy number (Kallioniemi et al., 1994, Proc. Natl. Acad. Sci. USA, 91:2156-60; Isola et al., 1995, Am. J. Path., 147: 905-11). These regions are predicted to encode dominantly acting genes that may play a role in tumor progression or response to therapy. Three of these regions have been associated with oncogenes: ERBB2 at 17q12, MYC at 8q24, and CCND1 and EMSI at 1q13. Amplification at 20q13, which occurs in a variety of tumor types, has not previously been associated with a known oncogene (Collins at al., 1998, Proc. Natl. Acad. Sci. USA, 95: 8703-08). The identification, cloning, and study of the genes involved in these chromosomal regions, e.g., 20q13, is crucial to the study of tumorigenesis and to the development of cancer diagnostics and therapeutics.
ADNF III is an activity dependent neurotrophic factor that has neurotrophic/neuroprotective activity as measured with in vitro cortical neuron culture assays described by, e.g., Hill et al., 1993, Brain Res. 603:222-233; Gozes et al., 1996, Proc. Natl. Acad. Sci. USA 93:427-432; Brenneman et al., 1988, Nature 335:636; Brenneman et al., 1990, Dev. Brain Res. 51:63; Forsythe and Westbrook, 1988, J. Physiol. Lond. 396:515. Previously, an ADNF III polypeptide has been shown to be involved in prevention of neuronal cell death (U.S. Ser. No. 60/037,404; PCT/US98/02485; U.S. Ser. No. 09/187,330) and ADNF III polypeptide have recently been shown to prevent conditions associated with fetal alcohol syndrome (U.S. Ser. No. 09/267,511). However, the ADNF III gene has not previously been shown to be involved in tumorigenesis, and has not been used to inhibit cancer cell growth.
It has been discovered that the ADNF III gene is unexpectedly located in a chromosomal region associated with cancer. Surprisingly, it has been found that ADNF III antisense oligonucleotides inhibit the growth of pathologically proliferating cells such as cancer cells. The present invention therefore provides methods and kits for inhibiting the proliferation of pathologically proliferating cells by using human ADNF III antisense oligonucleotides. The invention further provides methods and kits for detecting altered regulation of ADNF III in human cancers.
The methods and kits for inhibiting pathologically proliferating cells comprise the step of contacting the cells with an antisense oligonucleotide that is substantially complementary, preferably fully complementary, to a subsequence of an ADNF III nucleic acid. The ADNF III nucleic acid is preferably a human ADNF III nucleic acid, and preferably an ADNF III mRNA. The methods may also comprise the step of contacting the cells with two or more antisense oligonucleotides that are complementary to two or more different subsequences of an ADNF III nucleic acid. In one embodiment, the oligonucleotides used in these methods and kits are substantially complementary to a subsequence of the 5xe2x80x2 region of the ADNF III mRNA, and are preferably complementary to a subsequence of the ADNF III mRNA that encodes a methionine. In one embodiment, the oligonucleotides used in these methods are added to the pathologically proliferating cells at a concentration of 10 xcexcM.
The methods for detecting pathologically proliferating cells comprise the steps of comparing the level of ADNF III expression in a test cell with the level of ADNF III expression in a control cell, as determined by contacting the cells with a nucleic acid probe that is substantially complementary, preferably fully complementary, to a subsequence of an ADNF III mRNA, and identifying as a pathologically proliferating cell a cell in which ADNF III mRNA expression is at least twice the level of ADNF III mRNA expression in the control normal cell. The nucleic acid probes used in these methods preferably comprise a detectable moiety.
These methods and kits for inhibiting and detecting pathologically proliferating cells can be used to inhibit and/or detect the growth of malignant pathologically proliferating cells, such as breast cancer, neuroblastoma, ovarian cancer, endometrial cancer, prostate cancer, bladder cancer, lung cancer, esophageal cancer, neuroendocrine cancer, brain cancer, colon cancer, testicular cancer, pancreatic cancer, and leukemia cancer cells. These methods and kits can also be used to inhibit and/or detect the growth of benign pathologically proliferating cells such as restenotic plaques in vascular smooth muscle (atherosclerosis and restenosis), benign prostatic hyperplasia cells, retinal hemangioblastomas, and psoriatic cells.
The invention also provides kits for inhibiting.and detecting pathologically proliferating cells comprising nucleic acid probes that are substantially complementary to a subsequence of an ADNF III mRNA, preferably fully complementary to a subsequence of the ADNF III mRNA. The nucleic acid probes used in these kits preferably comprise a detectable moiety.
In one embodiment, the oligonucleotides and nucleic acid probes used in the methods and kits of the invention are selected from the group consisting of:
5xe2x80x2-TTGACAGGAAGTTGGAACAT-3xe2x80x2 (SEQ ID NO:1),
5xe2x80x2-GCTTCATAGGACTTTGGCAT-3xe2x80x2 (SEQ ID NO:2),
5xe2x80x2-ATCCTTGGTGGGAGTCCCAT-3xe2x80x2 (SEQ ID NO:3), and
5xe2x80x2-ACCTAGACCCAGTCTCAT-3xe2x80x2 (SEQ ID NO:6).
Preferably, the oligonucleotides and probes are fully complementary to an ADNF III mRNA, preferably to a human ADNF III mRNA. In one embodiment, the oligonucleotides are complementary to the 5xe2x80x2 region of the ADNF nucleic acid.
The antisense oligonucleotides of the invention may be ribozymes.
Additionally, the oligonucleotides and nucleic acid probes of the invention may be DNA oligonucleotides, peptide nucleic acid oligonucleotides, phosphorothioate oligonucleotides, or 2xe2x80x2-O methyl oligonucleotides. The oligonucleotides and probes are from about 8 to 50 nucleotides in length, and preferably about 15 to 25 nucleotides in length.